All reagents were provided from Dong-A ST (Yongin, Korea). DA-9601, the standardized ethanol extract of Artemisia asiatica, was supplied by Dong-A Pharmaceutical Co. Ltd. (Yongin, Korea). The percentage of eupatilin, a pharmacologically active ingredient of Artemisia asiatica, was found to be 2.25% (w/w) as determined by HPLC. DA-9601 was dissolved in 5 ml 3% hydroxypropyl methylcellulose (HPMC) (Sigma-Aldrich, St. Louis, MO, USA) solution.

Six-week-old male Sprague Dawley (SD) rats were obtained from Daehan Bio Link (Eumseong, Korea); those with body weights of approximately 200–250 g were used for the experiments. Animals were acclimated for a week and maintained at an ambient temperature of 23 ± 3°C with 12-h light/dark cycles (lights on 07:00–19:00). Food pellets (Daehan Bio Link) and water were provided ad libitum. After a 1-week adaptation period, rats were randomly assigned to five groups of 8–10 rats each. All animal procedures adhered to the National Institutes of Health Guidelines for Animal Research and were approved by the Dankook University Institutional Animal Care and Use Committee (IACUC; DKU-15-001), which adheres to the guidelines issued by the Institution of Laboratory Animal Resources.

Male SD rats weighing around 200–250 g were divided into five groups of 8–10 rats each. The first group was a control group treated with 3% HPMC for 4 days. The second group was pretreated with 3% HPMC, and ACF was then orally administered (50 mg/kg body weight) once a day for 2 days. The third group was pretreated with DA-9601 (100 mg/kg body weight) for 2 days and then co-treated with ACF (50 mg/kg) + DA-9601 (100 mg/kg) for 2 days. The fourth group was pretreated with esomeprazole (ESO) (10 mg/kg body weight) for 2 days and then co-treated with ACF (50 mg/kg) + ESO (10 mg/kg) for 2 days. The last group was pretreated with DA-9601 (100 mg/kg) + ESO (10 mg/kg) for 2 days and then co-treated with ACF (50 mg/kg) + DA-9601 (100 mg/kg) + ESO (10 mg/kg) for 2 days. Two-day consecutive dosing of ACF was selected from a preliminary study that measured small intestinal inflammatory lesions; in this study, treatment with 50 mg/kg of ACF for two consecutive days was observed to cause detectable ulcers in the small intestine. One-day dosing did not cause apparent inflammatory lesions in the small intestine. ACF dose of 100–200 mg/kg was observed to cause severe small intestinal inflammatory lesions and hence was not deemed appropriate for this study. Post 24 h of the last drug administration, the rats were euthanized by ether anesthesia. The small intestine, from the gastric duodenum to the large intestine, was quickly removed by incising the abdomen. The excised small intestine was washed with sterilized physiological saline to remove feces. Samples collected from each experimental group for the analysis of inflammatory mediators were stored at –80°C till further investigation.

The freshly excised small intestine was thoroughly washed with sterilized physiological saline to remove feces, and 1% paraformaldehyde was immediately added for fixation, for 30 min. Fixed tissues were spread out and visually observed. Inflammation lesions of the small intestine were mainly found in the lower intestine; hence, the lower part of the intestine was used for subsequent experiments. Data on the visual observation were collected using photography, and a quantitative analysis of the inflammation was performed using the following observation score criteria (Tables 1, 2). Small intestine tissue samples (1 cm × 1 cm) from experimental animals were fixed in 4% neutral buffered formaldehyde and processed by routine techniques prior to embedding in the paraffin blocks in the pathology lab at Dankook University Hospital. Briefly, a thick section (4 μm) was mounted on a glass slide and stained with hematoxylin and eosin. The sections were examined under a light microscope (CK-40; Olympus, Tokyo, Japan) by a pathologist who was blinded to the treatment.

Total RNA was isolated from the collected samples by using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). RNA was reverse transcribed into cDNA by using MMLV reverse transcriptase (Bioneer, Daejeon, Korea) and oligo-d(T)₁₈ primer. Quantitative real time PCR (qRT-PCR) was performed using a Rotor Gene SYBR Green Supermix Kit (Qiagen, Hilden, Germany), and the fluorescence was measured using a Rotor-gene PCR Cycler (Qiagen). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene. The primers used for qRT-PCR were synthesized by Cosmo Genetech (Seoul, Korea). The sequences of the forward and reverse primers used in the qRT-PCR and Semi-quantitative RT-PCR (sqRT-PCR) are as follows: IL-6 (275 bp), forward 5'-GGAGTTCCGTTTCTACCTGG-3' and reverse 5'-GCCGAGTAGACCTCATAGTG-3'; TNF-α (316 bp), forward 5'-CCACGTCGTAGCAAACCACCAAG-3' and reverse 5'-CAGGTACATGGGCTCATACC-3'; IL-17 (178 bp), forward 5_′-ACAGTGAAGGCAGCGGTACT-3_′ and reverse 5_′-GCTCAGAGTCCAGGGTGAAG-3_′; GAPDH (134 bp), forward 5_′-AACGGCACAGTCAAGGCTGA-3_′ and reverse 5_′-ACGCCAGTAGACTCCACGACAT-3_′. The relative expression levels of IL-6, IL-17, and TNF-α were measured as an index of gene expression related to inflammation. Three biologically independent experiments were performed, and each PCR reaction was repeated in triplicates. The relative level of a specific mRNA was calculated by normalizing its expression to that of GAPDH by using the 2^−ΔΔCt method. The sqRT-PCR reactions were performed using PCR PreMix (Bioneer). The PCR product of each gene was analyzed using 1.5% agarose gel electrophoresis, the bands were stained using Syto 60 (Li-COR Biosciences, Lincoln, NB, USA), and the stained gel was visualized using the Odyssey infrared imaging system (Li-COR Biosciences).

Samples were prepared according to the manufacturer’s instructions, and the chemokines or cytokines were quantified using an ELISA kit. Concentrations of IL-6, IL-1β, IL-10, GM-CSF, IFN-γ, and TNF-α in the small intestines of the drug-treated rats (50 mg/kg of ACE) were determined using an ELISA kit (Ab frontier, South Korea) following the manufacturer’s protocol. The concentration of whole homogeneous lysates obtained from each sample was normalized and the lysates were diluted within the measurable range. Optical density was measured at 450 nm (A450) using a microplate reader (Thermo Scientific Multiskan GO, Microplate Spectrophotometer; Thermo Fisher Scientific). Briefly, small intestine tissue (100 mg) was washed with 1X PBS, and then 1 ml of 1X PBS was added to completely homogenize the isolated small intestine by using a tissue disperser homogenizer. Homogenates were centrifuged at 5,000 g for 5 min at 2–8°C. The supernatant was used to measure the enzyme activity. To normalize the protein levels in all supernatant samples, the protein concentration was quantified in each sample by using the Lowry protein assay kit (Bio-Rad, Hercules, CA, USA).

The small intestine samples were washed 3 times with 1X PBS at 4°C, and then the intestinal epithelium was separated with cell separation solution at 37°C. The cell separation solution was composed of 10% fetal bovine serum, 20 mM Hank's balanced salt solution, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, 10 mM EDTA, and 10 μg/ml polymyxin B. After washing 5 times with 1X PBS, RPMI 1640 (Sigma-Aldrich) containing 400 U/ml collagenase D (Roche, Mannheim, Germany) and 10 μg/ml DNase I (Roche) was added along with 10% FBS, and the samples were incubated at 37°C for 90 min. Subsequently, 10 mM EDTA was added, and the samples were subjected to flow cytometry. The cells in the small intestine were analyzed by flow cytometry on the FACSCalibur system using the Cell Quest software (Becton Dickinson, Franklin Lakes, NJ, USA). The antibodies used were phycoerythrin (PE)-binding anti-mouse CD4 (GK1.5, Rat IgG2b, κ), peridinin-chlorophyll protein-binding anti-mouse CD4 (GK1.5), fluorescein isothiocyanate-binding anti-mouse IFN-γ (XMG1.2, Rat IgG1, κ), PE-binding anti-mouse IL-4, PE-binding anti-mouse IL-9 (RM9A4, Rat IgG1, κ), PE-binding anti-mouse IL-17A (TC11-18H10.1, Rat IgG1, κ), and Alexa Fluor 488 binding anti-mouse Foxp3 (150D, Mouse IgG1, κ) (Biolegend, San Diego, CA, USA). Fifty thousand single cells were analyzed per experiment.

All data are presented as the mean ± SEM. All statistical analyses were performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Student’s t-test was used for comparing the data between two groups, whereas one-way analysis of variance (ANOVA) was used for comparisons of data from more than three groups. The significance for all comparisons of interest was assessed, and p < 0.05 was considered statistically significant.

The severity of inflammation was evaluated using the observation score of inflammation (Tables 1, 2), and the experimental observations were blinded for maintaining the objectivity of the data. In the control group, no significant ulcers, necrosis, abscesses etc. were observed in the small intestine (Fig. 1A). When ACF (50 mg/kg) was administered for 2 days, many extensive ulcer sites were observed, and the inflammation score was counted as 5, which represents the most severe stage (Fig. 1B). Lesions were observed in the form of multifocal discrete small necrotic ulcers with intervening normal mucosa. The activities of the experimental animals slowed after ACF administration; however, no death was reported during the study. When DA-9601(100 mg/kg) was administered concurrently with ACF, significantly fewer ulcer sites than when ACF was administered alone were observed in the small intestine; the inflammation score tended towards 3–4 (Fig. 1C, E). However, when ESO (10 mg/kg) was administered concurrently with ACF, no significant change in the inflammation score as compared to that with the administration of ACF alone was observed (Fig. 1D).

The gene expression of inflammatory mediators such as IL-6, IL-17, and TNF-α in ACF-induced enteritis was analyzed. The expression of IL-6 was significantly increased by ACF administration compared to that in the controls. However, the expression of IL-6 was significantly decreased in the ACF + DA-9601 treated group as compared to that in the ACF treated group. Interestingly, the expression of IL-6 was found to have significantly increased in the group treated with ACF + ESO (Fig. 2A). The expression of IL-17 in the ACF treated group was significantly increased as compared to that in the control group; however, the expression of IL-17 was found to have significantly decreased in the ACF + DA-9601 treated group compared to that in the ACF treated group. The expression of IL-17 was the highest in the group treated with ACF + ESO (Fig. 2B). The expression of TNF-α in the ACF treated group was significantly increased compared to that in the control group. The expression of TNF-α was also significantly increased in the group treated with ACF + ESO as compared to the ACF treated group. However, the expression of TNF-α was found to have significantly decreased in the ACF + DA-9601 treated groups as compared to that in the ACF treated group (Fig. 2C). When ACF + ESO + DA-9601 was administered concurrently, a significant decrease in the expression of genes encoding for inflammatory mediators was observed as compared to the gene expression levels observed in the ACF + ESO treated group; nonetheless, the expression of these genes did not decrease as much as in the ACF + DA-9601 treated group. In particular, the expression of the gene encoding for TNF-α was significantly decreased in the group treated with ACF + ESO + DA-9601 as compared to that in the group treated with ACF + ESO; the expression level was still significantly higher than that in the group treated with ACF alone.

Unlike gene expression, the concentration of IL-6 in the small intestine was not significantly changed by ACF treatment. In addition, no change was induced by the ACF + DA-9601 treatment. However, the IL-6 concentration was significantly decreased in the ACF + ESO treated group; the level was restored to the control level by the ACF + ESO + DA-9601 combination treatment (Fig. 3A). The concentration of IL-10, an anti-inflammatory cytokine known as human cytokine synthesis inhibitory factor, was significantly decreased by ESO in ACF-induced enteritis but was significantly increased in the ACF + DA-9601 and ACF + ESO + DA-9601 treatment groups (Fig. 3B). In the ACF-induced enteritis, the concentrations of IL-1β and GM-CSF were significantly increased compared to control; however, when DA-9601 or ESO was concurrently applied with ACF, the concentrations of IL-1β and GM-CSF were significantly decreased. On the other hand, the concentrations of IL-1β and GM-CSF were found to have been significantly increased in the ACF + ESO + DA-9601 treated group (Fig. 3C, D). The concentrations of IFNs and TNF-α, which are crucial inflammatory cytokines, were found to have significantly increased in ACF-induced enteritis. However, both cytokines were significantly decreased in the ACF + DA-9601 and ACF + ESO treated groups. In addition, the concentrations of interferons (IFNs) and TNF-α were found to have significantly decreased by ACF + ESO + DA-9601 treatment (Fig. 3E, F). IFNs and TNF-α have been suggested as the crucial molecules in the inflammatory processes and are thought to influence cellular, tissue, and global physiological functions.

In inflammatory bowel disease, inflammatory mediators related to T cell imbalance and dysfunction are known to continuously activate the inflammatory response, thus causing chronic tissue damage [2]. The characteristics of CD4⁺ T cells in the small intestine were analyzed using flow cytometry (Fig. 4). Fig. 4A shows ‘dot plot’ analysis displaying target cell populations and Fig. 4B demonstrates relative percentage of target cell populations for easy understanding of the dot plots. IFN-γ⁺ CD4⁺ cells were significantly increased, compared to the control, in ACF-induced enteritis. There was no significant change in the ACF + DA-9601 treated group, but the fraction was found to have increased by ESO treatment. However, IFN-γ⁺ CD4⁺ cell fraction was observed to have decreased by the concurrent treatment of ACF + ESO + DA-9601 (Fig. 4B-a). IL-4⁺ CD4⁺ cells also increased in ACF-induced enteritis. These cells were reduced in the ACF + DA-9601 treated group, but significantly increased in the ACF + ESO treated group. Similar to IFN-γ⁺ CD4⁺ T cells, IL-4⁺ CD4⁺ cells decreased by the concurrent treatment with ACF + ESO + DA-9601 (Fig. 4B-b). IL-9⁺ CD4⁺ cells showed no significant change compared to the control in ACF-induced enteritis. These cells were found to have decreased in the ACF + DA-9601 and ACF + ESO + DA-9601 treated groups but increased upon treatment with ACF + ESO (Fig. 4B-c). IL-17⁺ CD4⁺ cells were increased in ACF-induced enteritis. No significant change was caused by ACF + DA-9601 treatment, but these cells were increased upon ACF + ESO treatment. Compared to the ACF + ESO treated group, IL-17⁺ CD4⁺ cells further decreased in the ACF + ESO + DA-9601 treated group (Fig. 4B-d). Regulatory T cells suppress the immune response; therefore, in animal models, regulatory T cells are known to be effective in treating enteritis. Foxp3⁺ CD4⁺ cells were found to have increased in ACF-induced enteritis. It was decreased in the ACF + DA-9601 group but increased in the ACF + ESO group. Compared to the ACF + ESO group, it was decreased in the ACF + ESO + DA-9601 group, and the level of Foxp3⁺ CD4⁺ cells returned to normal control levels (Fig. 4B-e).